CN101883842B - Forming cell structure with transient linker in cage - Google Patents
Forming cell structure with transient linker in cage Download PDFInfo
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- CN101883842B CN101883842B CN2008801186481A CN200880118648A CN101883842B CN 101883842 B CN101883842 B CN 101883842B CN 2008801186481 A CN2008801186481 A CN 2008801186481A CN 200880118648 A CN200880118648 A CN 200880118648A CN 101883842 B CN101883842 B CN 101883842B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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Abstract
In a method of forming a cellular structure, cells and a transient linker are supplied to a volume partially enclosed by a cage. The linker facilitates initial attachment of adjacent cells to form a cell aggregate. The cage defines distributed openings that are sized to retain the cell aggregate. A fluid comprising a cell culture medium is supplied to the volume. The fluid is withdrawn from the volume through the openings. Aggregated cells retained in the volume are cultured to form a cell structure. A cell culturing device is provided which comprises a conduit and a cage in the conduit. A fluid flows in the conduit. The fluid comprises the cells, the transient linker and the cell culture medium. The cage retains aggregated cells formed in the fluid, and defines distributed openings that allow the fluid to flow through.
Description
The cross reference of related application
The application requires to enjoy in the right of priority of the 60/960th, No. 743 U.S. Provisional Application of submitting on October 11st, 2007, and the full content of this application is incorporated herein by reference.
Technical field
The present invention relates to cyto-architectural formation, in particular to being used to form the cyto-architectural method and apparatus of three-dimensional (3D).
Background technology
Because three-dimensional (3D) cellularstructure can be than the 2D cell culture cell behavior in the analogue body closer, so it is very important.For example, the 3D vitro tissue model that in-vivo tissue is had high fidelity has important using value in the foundation of organizational engineering and pathologic model, and can use it for the coagulation efficiency and mechanism of studying and testing potential treatment preparation.
In routine techniques, form the difficulty of 3D cell culture for effective conveying of material (such as cell culture medium) is provided by the cyto-architectural interior region of 3D.Since normally come transportation of substances with perfusion by cellularstructure, therefore when cellularstructure has comparatively large vol, just more difficult by this cellularstructure transportation of substances.In some routine techniquess, in order to form the minicell structure, in the upholder of extracellular, for example being coated on as cellularstructure provides in the hydrogel or thin-layer matrix of 3D extracellular matrix (ECM) upholder with cell envelope.This extracellular upholder has formed the barrier of restriction to the cell delivery material.
Summary of the invention
Therefore, need in the environment that is conducive to by the cellularstructure transportation of substances, form cellularstructure.Have been found that with the fence in short-term linker (transient linker) and the fluid line and can effectively form the minicell structure.At first, the short-term linker connects cell, forming cell aggregate, but can not form restriction to cell or from the permanent barrier of cell delivery material.Fence has been kept the cell of assembling here but has been had distributed opening and has been beneficial to come transportation of substances by the different zones in the cellularstructure that forms.Fence can comprise that general alignment becomes a plurality of micro-cylinders of U-shaped figure, and wherein, the gap between adjacent cylinder allows the fluid traffic, but the size in gap can be kept the cell of gathering here.
According to an aspect of the present invention, it provides a kind of formation cyto-architectural method.The method comprises to the volume part (volume) that is partly centered on by fence provides cell and short-term linker.Linker is conducive to the initial adhesion of flanking cell, to form cell aggregate.Fence has consisted of distributed opening, and the size of opening can be kept cell aggregate here.The fluid that comprises cell culture medium partly is provided to this volume.Partly regain fluid by opening from this volume.Cultivation is retained in the gathering cell in this volume part, to form cellularstructure.Before partly providing fluid to this volume, cell suspension also can be dissolved in linker in the fluid in fluid.Can keep flowing by this volume fluid partly.The density of the cell in the fluid can be approximately 500 ten thousand to about 600 ten thousand cell/mL, and the concentration of the short-term linker in the fluid can be approximately 6 μ M to about 8 μ M.Described opening can for distributed, be beneficial to by cellularstructure perfusion cell culture medium.Fence can be arranged in the pipeline, and fluid this pipeline of can flowing through.Pipeline can comprise bottom and the relative sidewall that extends from the bottom.Fence can comprise from bottom extension and a plurality of thrusts between sidewall.This thrust can comprise micro-cylinder.This micro-cylinder general alignment can be become U-shaped figure.Gap between two adjacent micro-cylinders can be approximately 10 microns to approximately 50 microns.Linker can comprise the polymine main chain and with the hydrazide group of main chain bonding.The molecular weight of linker can be approximately 2000 dalton to about 20000 dalton.Cell can comprise HepG2 cell or rat marrow stem cell.Cell can comprise aldehyde radical.Cell can comprise to be modified and is formed the cell of aldehyde radical on the surface of the cell of modifying.Apply suction force by the downstream at distributed opening and can order about flowing of fluid.
According to another aspect of the present invention, it provides a kind of cell culture apparatus.This device comprises: pipeline; The fluid that in this pipeline, flows, this fluid comprises cell, short-term linker and cell culture medium, and this linker is conducive to the initial adhesion of flanking cell, to form cell aggregate; And for the fence of keeping here in the pipeline of the gathering cell that fluid forms, this fence has consisted of the distributed opening that allows flow to cross.Fence can comprise that general alignment becomes a plurality of thrusts of U-shaped figure.Thrust can comprise micro-cylinder.Pipeline can have bottom and the relative sidewall that extends from the bottom, and thrust can extend from the bottom.The density of the cell in the fluid can be approximately 500 ten thousand to about 600 ten thousand cell/mL, and the concentration of the short-term linker in the fluid can be approximately 6 μ M to about 8 μ M.Cell can be suspended in the fluid.Linker may be dissolved in the fluid.Linker can comprise the polymine main chain and with the hydrazide group of main chain bonding.The molecular weight of linker can be approximately 2000 dalton to about 20000 dalton.Cell can comprise aldehyde radical.Cell can comprise HepG2 cell or rat marrow stem cell.Apply suction force by the downstream at distributed opening and can order about flowing of fluid.
When the following description of looking back by reference to the accompanying drawings the specific embodiment of the present invention, for those of ordinary skills, it is clear that other aspects of the present invention and characteristics will become.
Description of drawings
Illustrated in the accompanying drawing of embodiments of the present invention in the mode with example only,
Fig. 1 is the skeleton view of the fluid line (illustrative embodiments of the present invention) that is used to form the cell cultures structure;
Fig. 2 and 3 is the fluid line plan view from above in use of Fig. 1;
Fig. 4 is the Confocal Images of the representative 3D cell aggregate that forms according to the embodiment of the present invention;
Fig. 5 is the scanning electron photomicrograph (SEM) of the representative 3D cell aggregate that forms according to the embodiment of the present invention;
The representative cyto-architectural transmitted light images of Fig. 6 in microfluidic channels, forming according to the embodiment of the present invention;
Fig. 7 is in the microfluidic channels of Fig. 6 but the transmitted light images of the control cells structure that forms under different conditions;
The representative cyto-architectural Confocal Images of Fig. 8 to 11 under different in flow rate, forming;
Figure 12 is the column diagram of the dependency of demonstration viable cell per-cent and flow velocity;
Figure 13 is the Confocal Images of the cell culture of perfusion HepG2 cell in the fluid channel that forms according to the embodiment of the present invention;
Figure 14 is the transmitted light images of the cell culture of Figure 13;
Figure 15 is the Confocal Images of the cell culture of perfusion rat marrow stem cell of former generation in the fluid channel that forms according to the embodiment of the present invention;
Figure 16 is the transmitted light images of the cell culture of Figure 15;
Figure 17 is used to form the schematic diagram of the optionally fluid line of cell culture for explanation, it is the example of embodiments of the present invention; And
Figure 18 is the skeleton view that is used to form another fluid line of cell culture, and it is the example of embodiments of the present invention.
Embodiment
Fig. 1,2 and 3 has shown the fluid line 100 that is used to form with the culturing cell structure, and this fluid line is the example of embodiments of the present invention.Fluid line 100 can form fluid means or be the interior part that is used for providing the fluid channel of this device.This device can have miscellaneous part or the feature that does not show in Fig. 1,2 and 3, be used for being provided at the function that special applications uses this device to need.
The shape of the fluid channel that is limited by pipeline 100 and large I are selected according to concrete application (shape and the size that comprise the cell cultures structure that will form).In order to form the minicell culture, the width of the fluid channel in the pipeline 100 and height can be less than 1mm.For example, in some embodiments, pipeline 100 usually can have height from approximately 50 microns to the about rectangular cross section of 500 microns variations.
According to concrete application, pipeline 100 can be formed by any suitable material.For example, bottom 102 and sidewall 104 (and top) can be made up to form by glass, plastics or polymer materials or its.The polymkeric substance that is fit to can comprise polycarbonate, polyacrylic acid, thick-photoresist material Resins, epoxy (for example, by the U.S., compound in the SU-8 series that the MicroChem company of Massachusetts produces), polyoxymethylene, polymeric amide, poly-terephthalic acid polytetramethylene glycol ester (polybutylenterephthalate), polyphenylene ether, polydimethylsiloxane (PDMS), polyester film, polyurethane(s), poly(vinylidene fluoride) (PVDF), PMMA (polymethylmethacrylate), fluorine polysiloxane or its composition or mixture.Use the polymerizable material that is fit to can form described polymkeric substance, suitable polymerizable material comprises monomer, consists of block or any suitable precursor molecule of oligopolymer.The different piece of pipeline 100 can be formed by identical or different material.
General alignment is become U-type figure and be arranged in the pipeline 100 with a plurality of micro-cylinders 110 that sidewall 104 keeps at a certain distance away.The opening end of U-type figure is towards entrance 106, and the partially enclosed end of this U-type figure is towards outlet 108.The gap that adjacent micro-cylinder is 110 can be approximately 10 microns to approximately 50 microns.Can select the size in gap so that the required rate of flooding that passes through this gap to be provided, this will further specify hereinafter.Micro-cylinder 110 can have any suitable shape of cross section.The width of micro-cylinder 110 can change, for example in 10 microns to 50 microns scope.Can select according to the required cellularstructure that will form the height of cylinder.In some embodiments, for example, height of column can be from approximately 10 microns extremely approximately 500 microns variations.In some embodiments, cylinder can extend beyond the overall height of pipeline 100.
Micro-cylinder can different materials forms by the material identical from pipeline 100 or with it.
Micro-cylinder 100 has consisted of partially enclosed volume part or the Growth of Cells district 112 of being delineated out by dotted line as among Fig. 1 and 2.
Not necessarily, cylinder 100 may extend into the top cover of pipeline 100.Perhaps, the top that the horizontal stripe (not shown) connects relative paired cylinder 110 can be set, and this horizontal stripe can keep at a certain distance away with the top cover of pipeline 100.
In one embodiment, bottom 102 is made by glass, and other parts of pipeline 100 that comprise sidewall 104, cylinder 110 and top cover are by identical polymer formation.Sidewall 104, cylinder 110 and top cover can be used as integral unit and form.In this case, cylinder 110 extends downward glass bottom 102 from top cover.The polymer moieties of pipeline 110 can form by casting.Mould can be the silicon mould of microfabrication.Polymer moieties can closely be connected to form the fluid channel of sealing with glass bottom 102.Polymkeric substance and glass can or use movably by permanent chemical bond, and fastening piece (such as clip) is attached to each other.
In another embodiment, pipeline 100 can be used as the whole complete unit that forms.This unit can be formed by plastics, polymkeric substance etc.
In one embodiment, can use polydimethylsiloxane (PDMS) to form pipeline 100.At first, form the silicon template by deep reaction ion etching.Then, casting PDMS material in this silicon template.Then can be with PDMS structure oxidation in oxygen plasma of casting, for example approximately 1 minute, so that PDMS structure and cover plate (not shown) Chemical bond such as cover glass.Before use can be with this device sterilization.
Such as better explanation, in use fluid 114 flow through pipeline 100 and Growth of Cells district 112 in Fig. 2 and 3.
Described cell culture medium can contain any suitable material that is useful on the used specific cells of cultivation, and this is that those skilled in the art can understand.For example, substratum can comprise Growth of Cells and cultivate nutrient substance needs or desirable.For example, cell culture medium can contain Da Erbaikeshi MEM (DMEM), minimum essential medium (MEM), F-10 nutrient substance mixture, F-12 nutrient substance mixture, the substratum (RPMI substratum) by Roswell Park Memorial Research Institute, Yi Si Kao Fushi improvement Dole's Becquerel substratum (IMDM), glucose, foetal calf serum (FCS), penicillin/streptomycin, CO
2, somatomedin or other materials.
Cell can be selected so that they can be inoculated in the Growth of Cells district 112 when fluid 114 is being flowed through pipeline 100.Cell 116 can be finishing so that they can assemble by the short-term linker.For example, cell 116 can be the cell that contains aldehyde radical or contain aldehyde radical through modifying.Cell 116 can by HepG2 cell (people's liver cell system), former generation rat marrow stem cell (BMSC), A549 people's pulmonary epithelial cells system, HeLa people's cervical cell system, human glioma cell be U87 and U251, former generation pig (pig) liver cell or the cell of other types modified.
In order to cultivate HepG2 and BMSC cell, cell culture medium can comprise DMEM, 10%FCS and glucose.For the HepG2 cell, the glucose content in the cell culture medium can be higher, for example, and about 4.5g/L; And for the BMSC cell, can be lower, about 1.0g/L for example.
In one embodiment, may need to prevent the secure adhesion of cell and wall surface, so that can be at the required flow velocity of pipeline 100 interior maintenances.Thereby by selecting can not avoid adhesion not occur firmly adhering to the cell of particular wall surface adhesion or by the rate of flow of fluid of regulating in the pipeline 100.
Described substratum can be osteogenic and can be prepared by the basic medium that contains 100nM dexamethasone, 0.05mM xitix 2-phosphoric acid ester and 10mM β-phospho-glycerol.
Select the short-term linker so that cell 116 can be connected at first so that their adhering to each other and formation cell aggregates 122.Can select linker directly contact and make cell aggregation and form the 3D cell aggregate setting up at iuntercellular.The linker molecule only temporarily is combined with cell 116 and can be separated from adherent cell after cell is adhering to each other.With the transformation period of the linker of cell surface adhesion can be in approximately 1 day to approximately 5 days scope.In one embodiment, the transformation period can be approximately 12 hours or shorter.
For example, for the cell with surperficial aldehyde radical, linker can contain the hydrazides end group, and this group can cause with the aldehyde radical reaction gathering of cell.In one embodiment, the hydrazides group can with main polymer chain conjugation or the bonding such as polymine (PEI) main chain.In different embodiments, can use other linear polymer linkers, tree-like linker, two step linkers (two-step linker) etc.Such as in following document, having disclosed some short-term linkers that are fit to: people such as Zhao, " Dendrimer hydraidesas multivalent transient inter-cellular linkers; " Biomaterials, 29 (2008) 3693-3702; With the people such as De Bank, " Surface engineering of living myoblasts via selective periodateoxidation, " Biotechnology and bioengineering, vol.81,2003, pp.800-808.
The short-term linker will be after cell have formed aggregate and cellular segregation.Suitable is that the short-term linker can not form permanent barrier at cell peripheral.This just allows to cell or from cell transportation of substances effectively.In addition, this secretes ECM so that cell is set up natural cell-cell interaction, and sets up the cell-matrix interaction, and this may be that the support of 3D iuntercellular needs.
Linker can be based on nontoxic lower molecular weight PEI.In one embodiment, the molecular weight of linker can be approximately 2000 dalton to about 20000 dalton.Primary amine groups on the PEI arm can be changed to generate hydrazides, hydrazides can with the cell surface of chemically modified on the reaction of aldehyde projection and assemble cell.Linker can retain in the cell surface short-term, and its transformation period is approximately 2 days.Linker can be selected so that anchorage-dependent cell (anchorage-dependent cell) produces their natural surroundings that is used for the 3D support and need not to mix the exogenous biomaterial that hinders potentially the material conveying.Like this, the ECM that is used for support of himself can be secreted and accumulate to cell.
Some cells need to by base plate supports or anchor on substrate could survive, Growth and reproduction, these cells are called as anchorage-dependent cell.For example, mammalian cell (primary cell and clone) be exactly rely on adherent.As long as anchorage-dependent cell has been assembled and has been confined in the Growth of Cells district, just can use easily in embodiments of the present invention these cells, they support by adjacent cell.Therefore, need not this cell is fixed on the outer matrix upholder such as gel.
The temperature of fluid can be remained on the level that is suitable for cultivating specific cells.In one embodiment, temperature can be approximately 37 ℃.Use heating unit (not shown) and temperature regulator (not shown) can be controlled the temperature in the pipeline 100.In some embodiments, can be with in the embedding fluid means that is provided with pipeline 100 thereon of well heater (not shown).
The control flow passes through the initial cell aggregate 118 that connects of TIP linker through the flow velocity of pipeline 100 so that cell 116 forms.For the viablity (viability) that keeps cell, should select flow (fluid-flow rate of per unit area) to reduce the surging force that cell is produced by fluid flow or minimize it, keep simultaneously the sufficient supplies to the cell culture medium of the gathering cell of being captured by micro-cylinder 110.In some embodiments, come flowing of actuating fluid just can reduce the impact flow effect if apply suction force by the downstream at the distributed opening that is consisted of by micro-cylinder 110.For example, can apply effective suction force by exporting 108, as using fluid pump, rather than apply impellent at entrance 106.
It should be understood that fluid can be inconsistent in pipeline 100 interior mobile local flow, and in the different zones in passage with also can be different when the different time.For example, when cell aggregate during in the 112 interior gathering of Growth of Cells district, the fluid that flows into Growth of Cells district 112 or the gap between the micro-cylinder 110 of flowing through may be along with timed deceleration.If keep constant by the overall flow rate of pipeline 100, the fluid that then flows out Growth of Cells district 112 may accelerate along with the time.Yet, at some embodiments with in using, still can by regulate through the overall flow rate of piping 100 flow is controlled in the Growth of Cells district 112 in or the gap of 110 of cylinders in material conveying or velocity of diffusion.
Can be understood that, higher flow can provide faster diffusion in the vitellarium, but this also may increase the shearing force to cell.Therefore, can regulating also, the optimization total flux realizes required balance.
Can make flow velocity or flow and other exercisable parameter optimizations, so that the cell aggregate that forms in position enough limits by cylinder 110 greatly, and enough little and prevent the obstruction of fluid channel.The size that can regulate cell aggregate by the cell density in the fluid and iuntercellular linker concentration.In some embodiments, cell density can be approximately 500 ten thousand to about 600 ten thousand cell/mL, and iuntercellular linker concentration can be approximately 6 μ M to about 8 μ M.In one embodiment, cell density can be approximately 600 ten thousand cell/mL, and iuntercellular linker concentration can be approximately 6 μ M.If cell density and linker concentration are too high, then at entrance 106 serious obstruction may appear.If cell density and linker concentration are too low, then cell aggregation is difficult to effectively capture cell too slowly in Growth of Cells district 112.
Select the gap length of 110 of micro-cylinders so that fluid 114 and individual cells 116 flow through, still in Growth of Cells district 112, keep cell aggregate 118 here.Have been found that in about 10 microns gaps to about 50 micrometer ranges and be suitable for some cells.
When fluid 114 is flowed through pipeline 110 and the gap by 110 of micro-cylinders, cell aggregate 118 continued growths and the final cellularstructure 120 that forms, this cellularstructure have usually by the position of micro-cylinder 110 and shape restriction and the shape and size consistent with Growth of Cells district 112.Can be understood that, micro-cylinder 110 can be used for the restrictive cell aggregate, with the cellularstructure of formation with its consistent size.
According to concrete application, cellularstructure 120 can be cultivated required for some time, for example mostly be most several weeks.Can adjust the content of input fluid 114 and cell culture medium in the different steps of formation and culturing process.
Can observe or monitor the Growth of Cells in the pipeline 100.For example, can take in the training period cell aggregate or cyto-architectural image.For this purpose, at least one side of pipeline 100 can be transparent.The image of cell can adopt any suitable technology to obtain, such as co-focusing imaging, transmitted light imaging, SEM etc.
For imaging, mark or other purposes, can be with cell dyeing, such as the immunostaining by actin filament dyeing, E-cadherin, Feng Kusa dyeing etc.By changing the inclusion of input fluid 114, comprise to input fluid 114 adding suitable coloring material, can dye in position.
Not necessarily recirculation of the cell culture medium that in cell cultivation process, uses.The closed circuit circulatory system (not shown) can be set and can circulate with the hyperchannel vibratory pump and transport the fluid of cell culture medium.
The representational cell aggregate that forms according to above-mentioned steps and cyto-architectural image and test-results are presented among Fig. 4 to 16, and further describe hereinafter.
Can be understood that, can utilize easily the opening of 110 of micro-cylinders or gap to come by the different zones perfusion culture base in the cellularstructure 120, carry thereby increase to this structure and the material in this structure.
Effectively, micro-cylinder 110 is used for keeping here gathering cell 118 in fluid 114 interior formation at fluid line 100 interior formation fence.
In other embodiments, can form different being used for and keep the fence of assembling cell here.For example, fence can be made up to form by cylinder, bar, line etc. or its.Fence can have more than one open side.Under any circumstance, the volume part is all partly centered on by fence.This volume part will limit basically by the plastidogenetic cyto-architectural shape of keeping here.Fence should limit distributed opening with permission flow warp, and is conducive to partly pour into material by this volume.Yet, regain the size of the opening of fluid process from fence and should be able to keep the gathering cell here at least.Distributed opening should be arranged on the plural side of fence.The shape of fence is set so that its volume that centers on partly has required shape.In some embodiments, as shown in Figure 1, fence can have open side.In other embodiments, fence can be all partly closed at least in all sides.The more than one side of fence can complete closed (referring to, the bottom 102 of pipeline 100 among Fig. 1 for example).Can be understood that, distributed opening that can be by partially enclosed side or the open side by fence partly provide cell, short-term linker and cell culture medium to the volume that crosses.In some embodiments, can partly provide input fluid to volume by the opening at fence bottom or top.
In some embodiments, fence can be formed by rigid material.In other embodiments, the part fence can be formed by flexible materials.For example, a side of fence can be formed by the flexible materials such as net or film.In some applications, when being subject to fluid flow and being retained in the extruding of the gathering cell in the fence, described net or film can have predetermined profile.
Fence can have the wholly or in part top of sealing.
Fence can form in pipeline or be arranged in the pipeline.Pipeline can have the shape of passage or chamber etc.Side with fence of distributed opening can keep at a certain distance away to allow fluid to pass through the effective traffic of opening with the wall of pipeline.Fence can immerse in the fluid that transports cell culture medium wholly or in part.
Can be understood that now, can improve in Fig. 1 to 3 arrangement that shows and some benefits or the advantage that still can realize mentioning in the application.
For example, Figure 17 has shown fluid line 200, and it has bottom 202, sidewall 204, entrance 106, outlet 208 and cylinder 210.As previously described fluid line 100, fluid 214 pipeline 200 of also flowing through.This like cell 218 forms cell aggregate 218 and the final cellularstructure 220 that forms.Entrance 206 links to each other with 3 input channels, i.e. middle pipeline 222 and the pipeline 224 of both sides.Similarly, outlet 208 links to each other with 3 output channels, i.e. the pipeline 226 of centre and the pipeline 228 of both sides.
In one embodiment, the length of pipeline 200 can be approximately 10mm, and width is about 0.6mm, and highly is about 0.1mm.Each micro-cylinder 210 can have the oval cross section of demonstration, and its major axis is about 0.03mm for about 0.5mm minor axis.Gap between adjacent cylinder can be that approximately 0.02mm is wide.Blind end and the distance between the open end of U-type figure can be approximately 0.2mm.For such arrangement, according to the size of cell, the cell density of inoculation in the fluid can be approximately 1,500,000 to about 10,000,000 cell/mL.Cell is larger, and best cell density can be lower.
Elliptic cylindrical may have advantage in some applications.Yet in different application or embodiment, cylinder can have other shape of cross section.
In use, middle pipeline 222 can be connected with the cell harvestor (not shown), is used for providing cell 216 to pipeline 200.The pipeline 224 of side can be connected with the substratum source, is used for providing substratum to pipeline 200.The short-term linker may be dissolved in the substratum.Can arrange 4 to the valve (not shown) at entrance 206, be used for the control fluid flow and carry different materials to pipeline 200.For example, can open at first the valve that is connected with the pipeline 222 of centre, then after enough cell is provided to pipeline 200, it be closed.
When providing cell to pipeline 200, can by the pipeline 228 of side, for example use the syringe pump (not shown), from pipeline 200 withdrawn fluid 214.In the process of culturing cell structure 220, can be from all pipelines 226 and 228 withdrawn fluid 214.
During other of the embodiment that shows in Fig. 1 changed, micro-cylinder 110 can be defined partially enclosed volume part and the fence structure of distributed opening replaces.For example, as shown in Figure 18, fence wall rather than cylinder with spaced slit or opening can be set in it.
The fluid line 300 that shows among Figure 18 is similar with the fluid line 100 of Fig. 1, and fluid line 300 has bottom 302, sidewall 304, entrance 306 and exports 308.Fence wall 310 is set replaces cylinder 110 to be used for keeping here the gathering cell.Distributed opening 312 is set so that substratum flows through wall 310 is interior.
In another embodiment, fence structure can comprise the sieve of fence sample or the filtration unit of other types, is used for keeping the gathering cell here when allowing fluid to be retracted by sieve or filtration unit.
Other thrusts that also can extend with the diapire from pipeline replace micro-cylinder.For example, can be with the WO 2006/052223 (people such as Yu, exercise question is " Cell Culture Device ", and on May 18th, 2006 is open) in disclose be used for make micro-cylinder arrangement and the technique improvement of minisize fluid device and be applied to form the device that is fit to that uses in embodiments of the present invention.The content of WO 2006/052223 that will be relevant with making fluid means is incorporated the application by reference into.
Pipeline or the passage of wherein placing fence can have different shapes and size in different embodiments.For example, sidewall 104 and nonessential be parallel.
In some embodiments, pipeline can have the shape of chamber.This chamber can have rectangle, cylindrical or spherical shape usually.Also can be with shape or big or small other fluidic components or the device of being arranged to hold except keeping fence of pipeline.The entrance and exit that pipeline can any desired location in chamber be set.Can also fluid inlet and outlet be set at for example wall of diapire, sidewall or roof to provide selectable or other inlet/outlet for the fluid traffic.
In order to promote to form cell aggregate with the short-term linker, cell can have or modified and have the reaction projection at cell surface.The short-term linker can have for the corresponding end group that reacts the projection reaction cell " being glued " together.Cell surface can be processed by enzyme and be modified by heredity or by chemically modified and the formation reaction projection.Such as in following document, having disclosed the example technique that is used for the modified cells surface: the people such as B.Kellam, " Chemical modification of mammalian cellsurfaces, " Chem.Soc.Rev., 2003, vol.32, pp.327-337; The people such as E.Saxon; " Chemical and biological strategies for engineering cell surface glycosylation, " Annu.Rev.cell.Dev.Biol., 2001, vol.17, pp.1-23; The people such as S-M.Ong, " Transientinter-cellular polymeric linker, " Biomaterials, 2007, vol.28, pp.3656-3667 (below be called " Ong ") incorporates the full content of these documents into the application with way of reference.
In Ong, disclosed the suitable technology that is used to form the TIP linker.Also can use other TIP linkers or be used to form the technology of TIP linker.
Embodiment discussed herein and improvement only do not have limit for illustration purpose.Other improvement also is fine.
Illustrative embodiments described here can be advantageously utilised in the multiple application.
For example, when being combined with short-term linker and micro-cylinder and arranging, can form fast 3D cellularstructure (as approximately in 5 minutes), and have accurate shape and size.Can relatively easily implement to form step.
Because do not support cell with nonvolatil extracellular matrix or massive material, so can be by the transportation of substances fully and effectively of the different zones in the cellularstructure.Because fluid is by the gap traffic between cylinder, therefore during forming cellularstructure or can pass through this cellularstructure continous pouring afterwards.Owing to there not being nonvolatil external bracing (for example hydrogel matrix of coated cell), and fluid flow is continuous, therefore makes it possible to the material such as oxygen and trophoplasm is transported to cell and walks material such as metabolic waste from cell delivery.
In the application-specific such as drug test or biological study, it may be useful that the material of improvement is carried for send biology preparation to cell structure.
Thereby produce natural microenvironment and can realize high-caliber bionics by making emiocytosis and gathering its oneself ECM, also need not to utilize angiopoietic perfusion culture in the analogue body and need not to add exogenous biomaterial.Potentially, more senior bionics can help to gather more anticipation results from cells in vivo reaction (for example drug test or biological study).
The miniature 3D cellularstructure that forms is according to the embodiment of the present invention compared with the maxicell structure and can be demonstrated many advantages.For example, miniature 3D cellularstructure size is tightr, can have higher handlability, faster and relevant analysis can be provided, and can reduce the reagent volume that needs in many application.
The application's embodiment can use the bionical microchip of using for drug test.Can form Various Tissues by in the microchannel, assembling one or more cell types.Use short-term iuntercellular linker to replace to make emiocytosis and assembling the natural ECM that is used for support for the permanent hydrogel that cell supports, rather than rely on exogenous biomaterial.Therefore this cellularstructure cell behavior in the analogue body better.
For example in drug test, a plurality of fluid channels can be set high throughput is provided.Thereby can connecting from the gradient generator, the microchannel can carry out simultaneously to the cellularstructure in the different microchannels drug test of a concentration range.The gradient generator can be designed to line style, contrary flexure type (sigmoidal) or exponential type, therefore the needs according to the user provide multi-functional.Such diversity may be useful for improving existing integrated fluid loop, thereby the high throughput of using for drug test provides effective processing.
The multiple fluid that can be linked in sequence chip carries out the method that the application describes.Different chips can be used for cultivating the different cell types that represent the different tissues analogue.Perhaps, can be at the different cell type of different cavity indoor cultivation that is connected by microfluidic channels on a kind of chip.Therapeutical agent can be by organizing analogue circulation and can detecting in system level their effect.Such arrangement is to being that potential benefit is arranged based on system response evaluation and test candidate therapeutic agent, and the grade of system response is higher than tissue reaction.Potentially, this arrangement can be used in many different minisize fluid devices.
With transparent pipeline material (for example glass bottom), the enough existing imaging forms of micron order 3D cell culture energy that form in the microchannel are with easily imaging of high resolving power.For example, cell culture can come imaging with phase contrast microscope, confocal laser scanning microscope, CLSM or two-photon laser microscope.This makes the real time imagery of cultured cells in physiological 3D microenvironment (for example, being used for the research of multiple dynamic cellular process, such as epithelial cell polarization, protein transportation, endocytosis, transcytosis, propagation, apoptosis etc.) become possibility.Particularly, it makes the High content screening (high-content screening) at a kind of cell of the 3D microenvironment of the multiple application that is used for comprising drug test become possibility.Embodiments of the present invention can make the mammiferous imaging of cultivating with 3D when control centers on the microenvironment fluid flow of cell become possibility.
Embodiments of the present invention also can be used for the foundation of the external model of various diseases, for example, and formation of cancer, liver and pulmonary fibrosis or virus infection.By introducing different cell types or paathogenic factor, can set up a series of models of the different steps that represents disease prognosis.Such model is useful to the study of disease potential mechanism that forms and the potential therapeutical agent that test is used for disease treatment.
Embodiment
Example I
Add NaIO by the cell suspension in test tube
4And with the mixture incubation that obtains 15 minutes, use NaIO
4Modify HepG2 cell (people's liver cell system) cell surface and in the projection of cell surface generation aldehyde.
With the cell suspension modified in substratum.Cell density is about 500 ten thousand to 600 ten thousand cell/mL.This substratum comprises DMEM, glucose, FCS and penicillin/streptomycin.
The TIP linker also is dissolved in the substratum.This TIP linker is the polymer molecule that a plurality of hydrazides that are connected with polymine (PEI) main chain form.Linker concentration be approximately 6 μ M to about 8 μ M.
At room temperature, make the microchannel of culture medium solution by showing among Figure 17 of containing cell and TIP linker.The size of microfluidic channels is 1cm (length) * 0.6mm (width) * 0.1mm (highly).This microfluidic channels has two entrances and an outlet.One row gap is the central part that the oval micro-cylinder of 0.03mm * 0.05mm of 0.02mm is positioned at microfluidic channels, and it has consisted of width is the resident interval of cell (vitellarium) of 0.2mm.
Solution passes through passage with different flow velocitys.Come actuating fluid to flow by the exit end pumping fluid in the microchannel.
Observe the cell aggregate that in the Growth of Cells district, forms.The Confocal Images that in Fig. 4, has shown representational cell aggregate.For the existence of the lip-deep linker of showed cell, use the linker of being combined with fluorescence dye.Linker in Fig. 4 is shown as the white border circular areas around cell.As shown in Figure 4, some in coated and these coated cells of many cell connected body molecules contact with each other, and this shows that linker has affected the support between three-dimensional cell.
In Fig. 5, shown the SEM image of cell aggregate, wherein had the short-term linker, but be invisible to the naked eye.
Approximately after 5 minutes, in the Growth of Cells district that is limited by micro-cylinder, formed cellularstructure.In Fig. 6, shown the representative cyto-architectural transmitted light images that under the flow velocity of about 0.03mL/h, in the microchannel, forms.
After finishing inoculation, make the medium flow that does not contain TIP linker and cell cross the fluid channel.Formed the bar shaped cellularstructure that cross section is generally rectangle.
Example II
Except the aggregation conditions difference, be identically formed the contrast cellularstructure with embodiment 1.Particularly, for given cell density and TIP linker concentration, this flow velocity is relatively slow.Fig. 7 has shown this cyto-architectural transmitted light images.What can see is that under these conditions, the gap between cylinder is stopped up by cell aggregate.As a result, the structure of formation does not have well-defined shape and size.
EXAMPLE III
Except changing flow velocity with the best perfusion flow velocity that is identified for specific device and cell tests sample, be identically formed cellularstructure with embodiment 1.
During with 1 day the chien shih cell culture medium be selected from 0.01,0.03,0.06 or the flow rate of 0.22mL/hr form various cellularstructures.
The structure that obtains with fluorescence activity dyeing (fluorescence viability staining) evaluation.Viable cell is dyeed by fluorexon AM.Dead cell is dyeed by propidium iodide (propidium iodide).
Fig. 8,9,10 and 11 shown respectively 0.01,0.03,0.06 and the flow velocity of 0.22mL/hr under the representational cyto-architectural Confocal Images that forms.
The viablity that has shown cell under different in flow rate among Figure 12.Can find out, for this specific arrangement and these cells, viablity the highest (approximately 80%) under the flow velocity of 0.03mL/hr.Under other flow velocitys, viablity is about 50%.
Be not limited to any specific theory, can be contemplated that under too high flow velocity, be applied to the reduction that high shear force on the cell or pressure can cause viablity; And under excessively low flow velocity, the deficiency that material (nutrient substance) is carried can cause the reduction of viablity.
EXAMPLE IV
With with example I in the similar step described use anchorage-dependent cell (HepG2 and rat bone stem cell) to form cellularstructure, wherein flow velocity is 0.03mL/hr, and cultivates 3 or 14 days.Observe with mostly being most the time in 2 weeks and in the microchannel, formed the 3D cellularstructure that is fully supported.
Dye to estimate the viablity of cell with fluorescence activity.Use the maintenance of the 3D form of actin filament dyeing evaluation structure.
After cultivating respectively 3 or 14 days, the result has shown the maintenance of excellent cell viablity and 3D form.
Figure 13 is the cyto-architectural Confocal Images of the plastidogenetic representativeness of HepG2 after cultivating 3 days.Figure 14 is the SEM image of the same cell structure in the microchannel.
Figure 15 is the cyto-architectural Confocal Images of the former plastidogenetic representativeness of generation rat BMSC after cultivating 14 days.Figure 16 is the SEM image of the same cell structure in the microchannel.
The Confocal Images (not shown) of the Actin muscle of sample cell structure dyeing has also shown the typical 3-D form that is distributed as of cortex Actin muscle in sample structure.
Those skilled in the art can understand hereinbefore other features, benefit and the advantage of the embodiment of NM the application's description from this specification sheets and accompanying drawing.
Certainly, above-mentioned embodiment only is never to be in order to limit in order to explain.Described embodiment easily forms, many changes of arrangement of parts, details and working order.This invention is intended in all such changes of the encompasses that is defined by the claims.
Claims (30)
1. one kind forms cyto-architectural method, and it comprises:
Partly provide cell and short-term linker to the volume that is partly centered on by fence, wherein said cell comprises aldehyde radical, and described short-term linker comprise for the hydrazides group of described aldehyde radical reaction to be conducive to the initial adhesion of flanking cell, forming cell aggregate, described fence has consisted of the distributed opening that its size can be kept described cell aggregate here;
The fluid that comprises cell culture medium partly is provided to described volume;
Partly regain described fluid by described opening from described volume; And
The gathering cell that cultivation is kept here in described volume part is to form cellularstructure.
2. method claimed in claim 1 wherein, before partly providing described fluid to described volume, is dissolved in described cell suspension in the described fluid in described fluid and with described linker.
3. method claimed in claim 1, it comprises that the described fluid that makes by described volume part keeps flowing.
4. method claimed in claim 3, wherein, the density of the described cell in described fluid is 500 ten thousand to 600 ten thousand cell/mL, and the concentration of the described short-term linker in described fluid is 6 μ M to 8 μ M.
5. each described method in the claim 1 to 4, wherein, described opening is distributed, to be conducive to pouring into described cell culture medium by described cellularstructure.
6. each described method in the claim 1 to 4 wherein, described fence is arranged in the pipeline, and described flow is through described pipeline.
7. method claimed in claim 6, wherein, described pipeline comprises bottom and the relative sidewall that extends from described bottom.
8. method claimed in claim 7, wherein, described fence comprises from described bottom and extending and a plurality of thrusts between described sidewall.
9. method claimed in claim 8, wherein, described thrust comprises micro-cylinder.
10. method claimed in claim 9 wherein, becomes U-type figure with described micro-cylinder general alignment.
11. method claimed in claim 10, wherein, the gap between two adjacent described micro-cylinders is 10 microns to 50 microns.
12. each described method in the claims 1 to 3, wherein, described linker comprise the polymine main chain and with the hydrazides group of described main chain bonding.
13. the described method of claim 12, wherein, the molecular weight of described linker is 2000 dalton to 20000 dalton.
14. method claimed in claim 4, wherein, described linker comprise the polymine main chain and with the hydrazides group of described main chain bonding.
15. the described method of claim 14, wherein, described cell comprises the cell that forms aldehyde radical through modifying on the surface of described modified cells.
16. the described method of claim 15, wherein, described cell comprises HepG2 cell or rat marrow stem cell.
17. claim 3 or method claimed in claim 4 wherein, apply suction force by the downstream at described distributed opening and order about the described mobile of described fluid.
18. a cell culture apparatus, it comprises:
Pipeline;
The fluid that in described pipeline, flows, described fluid comprises cell, short-term linker and cell culture medium, wherein said cell comprises aldehyde radical, and described short-term linker comprise for the hydrazides group of described aldehyde radical reaction to be conducive to the initial adhesion of flanking cell, to form cell aggregate; And
Fence in described pipeline, described fence are used for keeping the gathering cell that forms here in described fluid, and described fence has consisted of the big or small distributed opening that can keep described cell aggregate here and allow described flow warp.
19. the described device of claim 18, wherein, described fence comprises that general alignment becomes a plurality of thrusts of U-type figure.
20. the described device of claim 19, wherein, described thrust comprises micro-cylinder.
21. the described device of claim 19, wherein, described pipeline has bottom and the relative sidewall that extends from described bottom, and described thrust extends from described bottom.
22. each described device in the claim 18 to 21, wherein, the density of the described cell in described fluid is 500 ten thousand to 600 ten thousand cell/mL, and the concentration of the described short-term linker in described fluid is 6 μ M to 8 μ M.
23. each described device in the claim 18 to 21, wherein, with described cell suspension in described fluid.
24. each described device in the claim 18 to 21 wherein, is dissolved in described linker in the described fluid.
25. each described device in the claim 18 to 21, wherein, described linker comprise the polymine main chain and with the hydrazides group of described main chain bonding.
26. the described device of claim 25, wherein, the molecular weight of described linker is 2000 dalton to 20000 dalton.
27. the described device of claim 22, wherein, described linker comprise the polymine main chain and with the hydrazides group of described main chain bonding.
28. the described device of claim 27, wherein, described cell comprises the cell that forms aldehyde radical through modifying on the surface of described modified cells.
29. the described device of claim 28, wherein, described cell comprises HepG2 cell or rat marrow stem cell.
30. each described device in the claim 18 to 21 wherein, applies suction force by the downstream at described distributed opening and drives described fluid.
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| US96074307P | 2007-10-11 | 2007-10-11 | |
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| PCT/SG2008/000395 WO2009048435A1 (en) | 2007-10-11 | 2008-10-13 | Forming cell structure with transient linker in cage |
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| CN101883842A CN101883842A (en) | 2010-11-10 |
| CN101883842B true CN101883842B (en) | 2013-09-18 |
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| EP (1) | EP2203548A4 (en) |
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| EP2321405A4 (en) * | 2008-08-08 | 2012-09-12 | Agency Science Tech & Res | Microfluidic continuous flow device |
| JP5622189B2 (en) * | 2010-01-28 | 2014-11-12 | 国立大学法人東京工業大学 | Single cell separation plate |
| US9261496B2 (en) | 2010-09-29 | 2016-02-16 | Massachusetts Institute Of Technology | Device for high throughput investigations of multi-cellular interactions |
| WO2014071255A1 (en) * | 2012-11-01 | 2014-05-08 | The Charles Stark Draper Labortatory, Inc. | Ex vivo microfluidic analysis of biologic samples |
| WO2016200758A1 (en) * | 2015-06-10 | 2016-12-15 | Texas Tech University System | Microfluidic device for studying nematodes |
| EP3162890A1 (en) * | 2015-10-29 | 2017-05-03 | Shibuya Corporation | Method and apparatus for producing cell mass structure |
| KR102038622B1 (en) * | 2016-03-08 | 2019-10-30 | 삼성전자주식회사 | Slide for single cell detection and picking |
| CN108148750B (en) * | 2016-12-05 | 2021-10-15 | 中国科学院大连化学物理研究所 | Preparation method of multifunctional microfluidic chip for in-situ formation of embryoid body |
| EP3606669A1 (en) | 2017-04-03 | 2020-02-12 | The Charles Stark Draper Laboratory, Inc. | Microfluidic system for evaluation of chemotherapeutic and immunotherapeutic drugs |
| CA3059983C (en) | 2017-04-14 | 2023-09-05 | Advanced Solutions Life Sciences, Llc | Vascularized in vitro arrays of living cells |
| KR102048138B1 (en) * | 2018-01-31 | 2019-11-22 | 건양대학교산학협력단 | Separation device and alignment assembly of cells incubation piller |
| US10926261B1 (en) * | 2019-09-17 | 2021-02-23 | The Florida International University Board Of Trustees | Large microfluidic bioreactor and manufacturing method thereof |
| DE102019132214B3 (en) * | 2019-11-27 | 2021-04-29 | Cellbricks Gmbh | 3D scaffold made of biocompatible polymer and its production, colonized with biological cells |
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| Publication number | Publication date |
|---|---|
| WO2009048435A1 (en) | 2009-04-16 |
| JP2011500018A (en) | 2011-01-06 |
| US8389277B2 (en) | 2013-03-05 |
| US20100216241A1 (en) | 2010-08-26 |
| EP2203548A1 (en) | 2010-07-07 |
| EP2203548A4 (en) | 2014-01-15 |
| CN101883842A (en) | 2010-11-10 |
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